Studies of the hormonal regulation of inositol 1-phosphate synthase (EC5.5.1.4) in liver and testis have been extended. In testis, synthase activity, previously shown to be reduced after hypophysectomy, was partially restored by gonadotropins and completely by testosterone. Diethylstilbestrol blocked the gonadotropins but not testosterone. Testosterone had no effect on synthase in intact animals. We conclude that testosterone is the immediate regulator of synthase activity in testis. In liver, synthase activity was stimulated by T3 and T4 in the intact animal. Synthase activity, reduced in diabetic liver, was restored to normal by T3 and T4. We confirm our previous conclusion that thyroid hormones are the immediate regulators of synthase activity in the liver. Free inositol, derived from inositol 1-phosphate by a specific phosphatase present in excess in all tissues, was increased in testis by hypophysectomy and reduced to normal by both gonadotropins and testosterone. In the diabetic liver free inositol remained unchanged. Since the phosphatase was not subject to hormonal regulation, free inositol synthesis is also regulated by controls on synmthase. Differential utilization of inositol in liver and testis must then account for the differential response of the inositol pools in those organs.